U.S. patent number 9,254,001 [Application Number 13/095,277] was granted by the patent office on 2016-02-09 for tobacco-derived components and materials.
This patent grant is currently assigned to R.J. Reynolds Tobacco Company. The grantee listed for this patent is Michael Francis Dube, Anthony Richard Gerardi, Crystal Dawn Hege Byrd. Invention is credited to Michael Francis Dube, Anthony Richard Gerardi, Crystal Dawn Hege Byrd.
United States Patent |
9,254,001 |
Hege Byrd , et al. |
February 9, 2016 |
Tobacco-derived components and materials
Abstract
The invention provides a method of extracting and isolating
certain compounds from tobacco. The resulting isolate can include
more than 90% by weight of a given compound and can be used as a
flavor component for tobacco material used in smoking articles and
smokeless tobacco compositions. Exemplary compounds that may be
present in the isolate according to the invention include, but are
not limited to, solanone, neophytadiene, megastigmatrienone,
.beta.-damascenone, norsolanadione, cis-abienol,
.alpha.-cembratrienediol, .beta.-cembratrienediol, sucrose esters,
and lutein.
Inventors: |
Hege Byrd; Crystal Dawn
(Lexington, NC), Gerardi; Anthony Richard (Winston-Salem,
NC), Dube; Michael Francis (Winston-Salem, NC) |
Applicant: |
Name |
City |
State |
Country |
Type |
Hege Byrd; Crystal Dawn
Gerardi; Anthony Richard
Dube; Michael Francis |
Lexington
Winston-Salem
Winston-Salem |
NC
NC
NC |
US
US
US |
|
|
Assignee: |
R.J. Reynolds Tobacco Company
(Winston-Salem, NC)
|
Family
ID: |
46028215 |
Appl.
No.: |
13/095,277 |
Filed: |
April 27, 2011 |
Prior Publication Data
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Document
Identifier |
Publication Date |
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US 20120272976 A1 |
Nov 1, 2012 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A24B
15/26 (20130101); A24B 15/24 (20130101); C07C
35/36 (20130101); A23G 1/0016 (20130101); A24B
15/241 (20130101); C07C 35/21 (20130101); C07H
1/08 (20130101); A24B 13/00 (20130101); C07C
35/02 (20130101); A24B 15/30 (20130101); C07H
3/04 (20130101) |
Current International
Class: |
A24B
15/34 (20060101); A24B 15/30 (20060101); C07H
13/02 (20060101); A24B 15/24 (20060101) |
Field of
Search: |
;131/275,276,352 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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101 129 216 |
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Feb 2008 |
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CN |
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4 082896 |
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Mar 1992 |
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JP |
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5 186489 |
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Jul 1993 |
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JP |
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2006 0054786 |
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May 2006 |
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KR |
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Other References
Coleman, III et al. , "The Use of a Non-equilibrated Solid Phase
Microextraction Method to Quantitatively Determine the Off-notes in
Mint and other Essential Oils," J. Sci Food Agric, 2004, vol. 84,
pp. 1223-1228. cited by applicant .
Coleman, III et al., "Headspace Solid-Phase Microextraction
Analysis of Artificial Flavors," J. Sci Food Agric., 2005, vol. 85,
pp. 2645-2654. cited by applicant .
Demole, et al. "A Chemical Study of Burley Tobacco Flavour
(Nicotiana tabacum L.) Volatile to Medium-Volatile Constituents
(b.p. < 84.degree. /0.001 Torr," Helv. Chim. Acta, 1972, vol.
55, No. 6, pp. 1866-1882. cited by applicant .
Ishikawa et al., "Water-Soluble Constituents of Dill," Chem. Pharm.
Bull., 2002, vol. 50, No. 4, pp. 501-507. cited by applicant .
Johnson, et al. "The Structure, Chemistry, and Synthesis of
Solanone. A New Anomalous Terpenoid Ketone from Tobacco," J. Org.
Chem., 1965, vol. 30, No. 9, pp. 2918-2921. cited by applicant
.
Kandra, et al. "Studies of the Site and Mode of Biosynthesis of
Tobacco Trichome Exudate Components," Arch Biochem Biophys., 1988,
vol. 265, No. 2 pp. 425-432. cited by applicant .
Ochiai, Ph,D., "Take Two," Gerstel Solutions Worldwide, 2006, No.
6, pp. 17-19. cited by applicant .
Saito, et al. "Inhibitory effects of Cembratriene-4,6-diol
Derivative on the Induction of Epstein-Barr Virus Early Antigen by
12-O-Tetradecanoylphorbol-13-acetate," Agric. Biol. Chem., 1987,
vol. 51, No. 3, pp. 941-943. cited by applicant .
Severson, et al. "Isolation and Characterization of the Sucrose
Esters of the Cuticular Waxes of Green Tobacco Leaf," J. Agric.
Food Chem., 1985, vol. 33, No. 5 pp. 870-875. cited by applicant
.
Still, et al. "Rapid Chromatographic Technique for Preparative
Separations with Moderate Resolutions," J. Org. Chem., 1978, vol.
43, No. 14, pp. 2923-2925 cited by applicant .
Ohya et al., "Sucrose Estes from the Surface Lipids of Nicotiana
Cavicola," Pytochemistry, 1994, pp. 143-145, vol. 37, No. 1. cited
by applicant.
|
Primary Examiner: Sanders; James
Assistant Examiner: Stewart; Kimberly A
Attorney, Agent or Firm: Womble Carlyle Sandridge &
Rice, LLP
Claims
What is claimed:
1. A method of extracting and isolating compounds from plants of
the Nicotiana species, comprising: receiving a plant material of
the Nicotiana species; contacting the plant material with a solvent
for a time and under conditions sufficient to extract one or more
desired compounds from the plant material into the solvent;
separating the solvent containing the one or more desired compounds
from the extracted plant material; and purifying the solvent
containing the one or more desired compounds to provide an isolate
comprising at least about 75 percent by weight of the one or more
desired compounds, the one or more desired compounds being selected
from the group consisting of solanone, neophytadiene,
megastigmatrienone, .beta.-damascenone, norsolanadione,
cis-abienol, .alpha.-cembratrienediol, .beta.-cembratrienediol,
lutein, degradation products thereof, and mixtures thereof, wherein
the solvent is dry steam.
2. The method of claim 1, wherein the plant material of the
Nicotiana species is provided in green form.
3. The method of claim 1, wherein the plant material of the
Nicotiana species is provided in cured form.
4. The method of claim 1, wherein the plant material of the
Nicotiana species is in a form selected from the group consisting
of whole leaf, laminae, cut filler, volume expanded, stems,
cut-rolled stems, cut-puffed stems, reconstituted tobacco, and
particulate.
5. The method of claim 1, wherein the one or more desired compounds
are selected from the group consisting of solanone, neophytadiene,
megastigmatrienone, .beta.-damascenone, and norsolanadione.
6. The method of claim 1, wherein the contacting step further
comprises collecting a distillate.
7. The method of claim 6, wherein the distillate comprises a water
layer and an oily layer.
8. The method of claim 7, wherein the solvent containing the one or
more desired compounds is selected from the group consisting of the
oily layer, the water layer, and a waste stream generated from the
distillation process.
9. The method of claim 1, wherein the purifying step comprises
using preparative scale liquid chromatography.
10. The method of claim 1, wherein the purifying step comprises
using flash chromatography.
11. The method of claim 1, wherein the purifying step provides an
isolate comprising greater than about 90% by weight of the one or
more desired compounds.
12. The method of claim 1, wherein the purifying step provides an
isolate comprising greater than about 95% by weight of the one or
more desired compounds.
13. The method of claim 1, further comprising adding the isolate to
a tobacco composition adapted for use in a smoking article or a
smokeless tobacco composition.
14. The method of claim 1, further comprising treating the isolate
to provide one or more degradation products therefrom, wherein the
treating is selected from oxidation, heat treatment, and a
combination thereof.
15. A method of extracting and isolating compounds from plants of
the Nicotiana species, comprising: receiving a plant material of
the Nicotiana species; contacting the plant material with a solvent
for a time and under conditions sufficient to extract one or more
desired compounds from the plant material into the solvent;
separating the solvent containing the one or more desired compounds
from the extracted plant material; purifying the solvent containing
the one or more desired compounds to provide an isolate comprising
at least about 75 percent by weight of the one or more desired
compounds, the one or more desired compounds being selected from
the group consisting of solanone, neophytadiene,
megastigmatrienone, .beta.-damascenone, norsolanadione,
cis-abienol, .alpha.-cembratrienediol, .beta.-cembratrienediol,
lutein, degradation products thereof, and mixtures thereof; and
treating the isolate to provide one or more degradation products
therefrom, wherein the treating is selected from oxidation, heat
treatment, and a combination thereof, wherein the isolate comprises
lutein and wherein the one or more degradation products are
selected from the group consisting of megastigmatrienone,
beta-damascenone, and mixtures thereof, or wherein the isolate
comprises cis-abienol and wherein the one or more degradation
products are selected from the group consisting of sclareolide,
sclareol, ambroxide, and mixtures thereof.
16. The method of claim 15, wherein the plant material of the
Nicotiana species is in a form selected from the group consisting
of whole leaf, laminae, cut filler, volume expanded, stems,
cut-rolled stems, cut-puffed stems, reconstituted tobacco, and
particulate.
17. The method of claim 15, wherein the plant material of the
Nicotiana species is provided in green form.
18. The method of claim 15, wherein the plant material of the
Nicotiana species is provided in cured form.
19. The method of claim 15, wherein the solvent is methanol.
20. The method of claim 15, wherein the purifying step comprises
using preparative scale liquid chromatography.
21. The method of claim 15, wherein the purifying step comprises
using flash chromatography.
22. The method of claim 15, wherein the purifying step provides an
isolate comprising greater than about 90% by weight of the one or
more desired compounds.
23. The method of claim 15, wherein the purifying step provides an
isolate comprising greater than about 95% by weight of the one or
more desired compounds.
24. The method of claim 13, wherein the isolate is added in the
form of a casing formulation or a top dressing formulation applied
to tobacco strip or in the form of a component of a reconstituted
tobacco material.
25. The method of claim 13, wherein the tobacco composition
comprises a tobacco material adapted for use in a smoking
article.
26. The method of claim 25, wherein the amount of isolate in the
tobacco composition is between about 5 ppm and about 5 weight
percent based on the total dry weight of the tobacco material in
the smoking article.
27. The method of claim 13, wherein the tobacco composition
comprises a tobacco material adapted for use in a smokeless tobacco
product.
28. The method of claim 27, wherein the amount of isolate in the
tobacco composition is between about 5 ppm and about 5 weight
percent based on the total dry weight of the tobacco material in
the smokeless tobacco product.
29. The method of claim 15, further comprising: adding the isolate
to a tobacco composition adapted for use in a smoking article or a
smokeless tobacco composition.
30. The method of claim 29, wherein the isolate is added in the
form of a casing formulation or a top dressing formulation applied
to tobacco strip or in the form of a component of a reconstituted
tobacco material.
31. The method of claim 29, wherein the tobacco composition
comprises a tobacco material adapted for use in a smoking
article.
32. The method of claim 31, wherein the amount of isolate in the
tobacco composition is between about 5 ppm and about 5 weight
percent based on the total dry weight of the tobacco material in
the smoking article.
33. The method of claim 29, wherein the tobacco composition
comprises a tobacco material adapted for use in a smokeless tobacco
product.
34. The method of claim 33, wherein the amount of isolate in the
tobacco composition is between about 5 ppm and about 5 weight
percent based on the total dry weight of the tobacco material in
the smokeless tobacco product.
Description
FIELD OF THE INVENTION
The present invention relates to products made or derived from
tobacco, or that otherwise incorporate tobacco, and are intended
for human consumption. Of particular interest are ingredients or
components obtained or derived from plants or portions of plants
from the Nicotiana species.
BACKGROUND OF THE INVENTION
Popular smoking articles, such as cigarettes, have a substantially
cylindrical rod shaped structure and include a charge, roll or
column of smokable material such as shredded tobacco (e.g., in cut
filler form) surrounded by a paper wrapper thereby forming a
so-called "tobacco rod." Normally, a cigarette has a cylindrical
filter element aligned in an end-to-end relationship with the
tobacco rod. Typically, a filter element comprises plasticized
cellulose acetate tow circumscribed by a paper material known as
"plug wrap." Certain cigarettes incorporate a filter element having
multiple segments, and one of those segments can comprise activated
charcoal particles. Typically, the filter element is attached to
one end of the tobacco rod using a circumscribing wrapping material
known as "tipping paper." It also has become desirable to perforate
the tipping material and plug wrap, in order to provide dilution of
drawn mainstream smoke with ambient air. A cigarette is employed by
a smoker by lighting one end thereof and burning the tobacco rod.
The smoker then receives mainstream smoke into his/her mouth by
drawing on the opposite end (e.g., the filter end) of the
cigarette.
The tobacco used for cigarette manufacture is typically used in
blended form. For example, certain popular tobacco blends, commonly
referred to as "American blends," comprise mixtures of flue-cured
tobacco, burley tobacco and Oriental tobacco, and in many cases,
certain processed tobaccos, such as reconstituted tobacco and
processed tobacco stems. The precise amount of each type of tobacco
within a tobacco blend used for the manufacture of a particular
cigarette brand varies from brand to brand. However, for many
tobacco blends, flue-cured tobacco makes up a relatively large
proportion of the blend, while Oriental tobacco makes up a
relatively small proportion of the blend. See, for example, Tobacco
Encyclopedia, Voges (Ed.) p. 44-45 (1984), Browne, The Design of
Cigarettes, 3.sup.rd Ed., p. 43 (1990) and Tobacco Production,
Chemistry and Technology, Davis et al. (Eds.) p. 346 (1999).
Tobacco also may be enjoyed in a so-called "smokeless" form.
Particularly popular smokeless tobacco products are employed by
inserting some form of processed tobacco or tobacco-containing
formulation into the mouth of the user. Various types of smokeless
tobacco products are set forth in U.S. Pat. No. 1,376,586 to
Schwartz; U.S. Pat. No. 3,696,917 to Levi; U.S. Pat. No. 4,513,756
to Pittman et al.; U.S. Pat. No. 4,528,993 to Sensabaugh, Jr. et
al.; U.S. Pat. No. 4,624,269 to Story et al.; U.S. Pat. No.
4,987,907 to Townsend; U.S. Pat. No. 5,092,352 to Sprinkle, III et
al.; and U.S. Pat. No. 5,387,416 to White et al.; US Pat. Appl.
Pub. Nos. 2005/0244521 to Strickland et al. and 2009/0293889 to
Kumar et al.; PCT WO 04/095959 to Arnarp et al.; PCT WO 05/063060
to Atchley et al.; PCT WO 05/004480 to Engstrom; PCT WO 05/016036
to Bjorkholm; and PCT WO 05/041699 to Quinter et al., each of which
is incorporated herein by reference. See, for example, the types of
smokeless tobacco formulations, ingredients, and processing
methodologies set forth in U.S. Pat. No. 6,953,040 to Atchley et
al. and U.S. Pat. No. 7,032,601 to Atchley et al., each of which is
incorporated herein by reference.
One type of smokeless tobacco product is referred to as "snuff."
Representative types of moist snuff products, commonly referred to
as "snus," have been manufactured in Europe, particularly in
Sweden, by or through companies such as Swedish Match AB, Fiedler
& Lundgren AB, Gustavus AB, Skandinavisk Tobakskompagni A/S,
and Rocker Production AB. Snus products available in the U.S.A.
have been marketed under the tradenames Camel Snus Frost, Camel
Snus Original and Camel Snus Spice by R.J. Reynolds Tobacco
Company. See also, for example, Bryzgalov et al., 1N1800 Life Cycle
Assessment, Comparative Life Cycle Assessment of General Loose and
Portion Snus (2005). In addition, certain quality standards
associated with snus manufacture have been assembled as a so-called
GothiaTek standard. Representative smokeless tobacco products also
have been marketed under the tradenames Oliver Twist by House of
Oliver Twist A/S; Copenhagen, Skoal, SkoalDry, Rooster, Red Seal,
Husky, and Revel by U.S. Smokeless Tobacco Co.; "taboka" by Philip
Morris USA; Levi Garrett, Peachy, Taylor's Pride, Kodiak, Hawken
Wintergreen, Grizzly, Dental, Kentucky King, and Mammoth Cave by
Conwood Company, LLC; and Camel Orbs, Camel Sticks, and Camel
Strips by R.J. Reynolds Tobacco Company.
Through the years, various treatment methods and additives have
been proposed for altering the overall character or nature of
tobacco materials utilized in tobacco products. For example,
additives or treatment processes have been utilized in order to
alter the chemistry or sensory properties of the tobacco material,
or in the case of smokable tobacco materials, to alter the
chemistry or sensory properties of mainstream smoke generated by
smoking articles including the tobacco material. The sensory
attributes of cigarette smoke can be enhanced by incorporating
flavoring materials into various components of a cigarette.
Exemplary flavoring additives include menthol and products of
Maillard reactions, such as pyrazines, aminosugars, and Amadori
compounds. See also, Leffingwell et al., Tobacco Flavoring for
Smoking Products, R.J. Reynolds Tobacco Company (1972), which is
incorporated herein by reference. In some cases, treatment
processes involving the use of heat can impart to the processed
tobacco a desired color or visual character, desired sensory
properties, or a desired physical nature or texture. Various
processes for preparing flavorful and aromatic compositions for use
in tobacco compositions are set forth in U.S. Pat. No. 3,424,171 to
Rooker; U.S. Pat. No. 3,476,118 to Luttich; U.S. Pat. No. 4,150,677
to Osborne, Jr. et al.; U.S. Pat. No. 4,986,286 to Roberts et al.;
U.S. Pat. No. 5,074,319 to White et al.; U.S. Pat. No. 5,099,862 to
White et al.; U.S. Pat. No. 5,235,992 to Sensabaugh, Jr.; U.S. Pat.
No. 5,301,694 to Raymond et al.; U.S. Pat. No. 6,298,858 to
Coleman, III et al.; U.S. Pat. No. 6,325,860 to Coleman, III et
al.; U.S. Pat. No. 6,428,624 to Coleman, III et al.; U.S. Pat. No.
6,440,223 to Dube et al.; U.S. Pat. No. 6,499,489 to Coleman, III;
and U.S. Pat. No. 6,591,841 to White et al.; US Pat. Appl.
Publication Nos. 2004/0173228 to Coleman, III and 2010/0037903 to
Coleman, III et al., each of which is incorporated herein by
reference.
The sensory attributes of smokeless tobacco can also be enhanced by
incorporation of certain flavoring materials. See, for example, US
Pat. Appl. Pub. Nos. 2002/0162562 to Williams; 2002/0162563 to
Williams; 2003/0070687 to Atchley et al.; 2004/0020503 to Williams,
2005/0178398 to Breslin et al.; 2006/0191548 to Strickland et al.;
2007/0062549 to Holton, Jr. et al.; 2007/0186941 to Holton, Jr. et
al.; 2007/0186942 to Strickland et al.; 2008/0029110 to Dube et
al.; 2008/0029116 to Robinson et al.; 2008/0029117 to Mua et al.;
2008/0173317 to Robinson et al.; and 2008/0209586 to Neilsen et
al., each of which is incorporated herein by reference.
There is a need in the art for flavorful compositions suitable for
addition to smoking products or smokeless tobacco products to
introduce desired characteristics. It would be desirable to provide
a method for efficient extraction and isolation of such
compositions.
SUMMARY OF THE INVENTION
The present invention provides a method of extracting and isolating
various compounds from plants of the Nicotiana species. The method
of the invention is selective for certain compounds that impart
flavorful characteristics and/or compounds that degrade to produce
compounds that impart flavorful characteristics to smoking articles
and/or smokeless tobacco products. The invention also provides
methods for processing these compounds and tobacco materials
incorporating these compounds.
Thus, in one aspect, the present invention provides a method of
extracting and isolating compounds from plants of the Nicotiana
species. In certain embodiments, the method comprises receiving a
plant material of the Nicotiana species; contacting the plant
material with a solvent for a time and under conditions sufficient
to extract one or more desired compounds from the plant material
into the solvent; separating the solvent containing the one or more
desired compounds from the extracted plant material; and purifying
the solvent containing the one or more desired compounds to provide
an isolate. In some embodiments, the isolate thus obtained
comprises at least about 75 percent by weight of the one or more
desired compounds. In certain embodiments, the one or more desired
compounds are selected from the group consisting of solanone,
neophytadiene, megastigmatrienone, .beta.-damascenone,
norsolanadione, cis-abienol, .alpha.-cembratrienediol,
.beta.-cembratrienediol, sucrose esters, lutein, degradation
products thereof, and mixtures thereof.
In some embodiments, the plant material of the Nicotiana species is
in a form selected from the group consisting of whole leaf,
laminae, cut filler, volume expanded, stems, cut-rolled stems,
cut-puffed stems, reconstituted tobacco, and particulate. In some
embodiments, the plant material of the Nicotiana species is
provided in green form or in cured form.
In certain embodiments, the solvent is methanol. The one or more
desired compounds thus obtained can be, for example, selected from
the group consisting of cis-abienol, .alpha.-cembratrienediol,
.beta.-cembratrienediol, sucrose esters, and lutein. In certain
embodiments, the solvent is dry steam. The one or more desired
compounds thus obtained can be, for example, selected from the
group consisting of solanone, neophytadiene, megastigmatrienone,
.beta.-damascenone, and norsolanadione. When the solvent is dry
steam, the contacting step can further comprise collecting a
distillate. In some embodiments, the distillate can comprise a
water layer and an oily layer and the solvent containing the one or
more desired compounds can be selected from the group consisting of
the oily layer, the water layer, and a waste stream generated from
the distillation process.
In some embodiments, the purifying step comprises using preparative
scale liquid chromatography. In certain embodiments, the purifying
step comprises using flash chromatography. The purifying step can
provide an isolate with a desired level of the one or more desired
compounds, for example, greater than about 90% by weight or greater
than about 95% by weight of the one or more desired compounds. In
certain embodiments, the method further comprises adding the
isolate to a tobacco composition adapted for use in a smoking
article or a smokeless tobacco composition.
In some embodiments, the isolate can be further treated to provide
one or more degradation products therefrom, wherein the treating
comprises oxidation (i.e., treating with H.sub.2O.sub.2 or another
oxidizing reagent) and/or heat treatment. As one example, the
isolate can comprise lutein and the one or more degradation
products can be selected from the group consisting of
megastigmatrienone, beta-damascenone, and mixtures thereof. As
another example, the isolate can comprise cis-abienol and the one
or more degradation products can be selected from the group
consisting of sclareolide, sclareol, ambroxide, and mixtures
thereof.
In another aspect of the present invention is provided a method for
providing a flavor material derived from a plant of the Nicotiana
species for addition to a tobacco composition, the method
comprising receiving a plant material of the Nicotiana species;
contacting the plant material with a solvent for a time and under
conditions sufficient to extract one or more desired compounds from
the plant material into the solvent; separating the solvent
containing the one or more desired compounds from the extracted
plant material; purifying the solvent containing the one or more
compounds to provide an isolate comprising at least about 75% by
weight of the one or more desired compounds, the one or more
desired compounds being selected from the group consisting of
solanone, neophytadiene, megastigmatrienone, .beta.-damascenone,
norsolanadione, cis-abienol, .alpha.-cembratrienediol,
.beta.-cembratrienediol, sucrose esters, lutein, degradation
products thereof, and mixtures thereof; and adding the isolate to a
tobacco composition adapted for use in a smoking article or a
smokeless tobacco composition.
Various modifications can be made to the method of providing a
flavor material, as noted above for the method of extracting and
isolating compounds. For example, the plant material and solvent
used can be varied, and various additional treatment methods can be
used in combinations with the inventive method.
The isolate can be added to the tobacco composition in a variety of
ways. For example, the isolate can be added the form of a casing
formulation or a top dressing formulation applied to tobacco strip
or in the form of a component of a reconstituted tobacco material.
In certain embodiments, the tobacco composition comprises a tobacco
material adapted for use in a smoking article. In such embodiments,
the amount of isolate in the tobacco composition can be, for
example, between about 5 ppm and about 5 percent by weight, based
on the total dry weight of the tobacco material in the smoking
article. In certain embodiments, the tobacco composition comprises
a tobacco material adapted for use in a smokeless tobacco product.
In such embodiments, the amount of isolate in the tobacco
composition can be, for example, between about 5 ppm and about 5
percent by weight, based on the total dry weight of the tobacco
material in the smokeless tobacco product.
In another aspect of the present invention is provided an isolate
from a plant of the Nicotiana species or components thereof,
wherein the isolate comprises at least about 75 percent by weight
of a compound selected from the group consisting of solanone,
neophytadiene, megastigmatrienone, .beta.-damascenone,
norsolanadione, cis-abienol, .alpha.-cembratrienediol,
.beta.-cembratrienediol, sucrose esters, lutein, and degradation
products thereof, and mixtures thereof. In some embodiments, the
isolate comprises greater than about 90% by weight or greater than
about 95% by weight of the one or more desired compounds. In some
embodiments are provided tobacco compositions comprising the
isolate for use in smoking articles or smokeless tobacco
compositions.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
The present invention now will be described more fully hereinafter.
This invention may, however, be embodied in many different forms
and should not be construed as limited to the embodiments set forth
herein; rather, these embodiments are provided so that this
disclosure will be thorough and complete, and will fully convey the
scope of the invention to those skilled in the art. As used in this
specification and the claims, the singular forms "a," "an," and
"the" include plural referents unless the context clearly dictates
otherwise. Reference to "dry weight percent" or "dry weight basis"
refers to weight on the basis of dry ingredients (i.e., all
ingredients except water).
The selection of the plant from the Nicotiana species can vary; and
in particular, the types of tobacco or tobaccos may vary. Tobaccos
that can be employed include flue-cured or Virginia (e.g., K326),
burley, sun-cured (e.g., Indian Kurnool and Oriental tobaccos,
including Katerini, Prelip, Komotini, Xanthi and Yambol tobaccos),
Maryland, dark, dark-fired, dark air cured (e.g., Passanda, Cubano,
Jatin and Bezuki tobaccos), light air cured (e.g., North Wisconsin
and Galpao tobaccos), Indian air cured, Red Russian and Rustica
tobaccos, as well as various other rare or specialty tobaccos.
Descriptions of various types of tobaccos, growing practices and
harvesting practices are set forth in Tobacco Production, Chemistry
and Technology, Davis et al. (Eds.) (1999), which is incorporated
herein by reference. Various representative types of plants from
the Nicotiana species are set forth in Goodspeed, The Genus
Nicotiana, (Chonica Botanica) (1954); U.S. Pat. No. 4,660,577 to
Sensabaugh, Jr. et al.; U.S. Pat. No. 5,387,416 to White et al. and
U.S. Pat. No. 7,025,066 to Lawson et al.; US Patent Appl. Pub. Nos.
2006/0037623 to Lawrence, Jr. and 2008/0245377 to Marshall et al.;
each of which is incorporated herein by reference. Exemplary
Nicotiana species include are N. tabacum, N. rustica, N. alata, N.
arentsii, N. excelsior, N. forgetiana, N. glauca, N. glutinosa, N.
gossei, N. kawakamii, N. knightiana, N. langsdorffi, N. otophora,
N. setchelli, N. sylvestris, N. tomentosa, N. tomentosiformis, N.
undulata, N. x sanderae, N. africana, N. amplexicaulis, N.
benavidesii, N. bonariensis, N. debneyi, N. longiflora, N.
maritina, N. megalosiphon, N. occidentalis, N. paniculata, N.
plumbaginifolia, N. raimondii, N. rosulata, N. simulans, N.
stocktonii, N. suaveolens, N. umbratica, N. velutina, N.
wigandioides, N. acaulis, N. acuminata, N. attenuata, N.
benthamiana, N. cavicola, N. clevelandii, N. cordifolia, N.
corymbosa, N. fragrans, N. goodspeedii, N. linearis, N. miersii, N.
nudicaulis, N. obtusifolia, N. occidentalis subsp. Hersperis, N.
pauciflora, N. petunioides, N. quadrivalvis, N. repanda, N.
rotundifolia, N. solanifolia and N. spegazzinii.
Nicotiana species can be derived using genetic-modification or
crossbreeding techniques (e.g., tobacco plants can be genetically
engineered or crossbred to increase or decrease production of
components, characteristics or attributes). See, for example, the
types of genetic modifications of plants set forth in U.S. Pat. No.
5,539,093 to Fitzmaurice et al.; U.S. Pat. No. 5,668,295 to Wahab
et al.; U.S. Pat. No. 5,705,624 to Fitzmaurice et al.; U.S. Pat.
No. 5,844,119 to Weigl; U.S. Pat. No. 6,730,832 to Dominguez et
al.; U.S. Pat. No. 7,173,170 to Liu et al.; U.S. Pat. No. 7,208,659
to Colliver et al. and U.S. Pat. No. 7,230,160 to Benning et al.;
US Patent Appl. Pub. No. 2006/0236434 to Conkling et al.; and PCT
WO2008/103935 to Nielsen et al.
For the preparation of smokeless and smokable tobacco products, it
is typical for harvested plants of the Nicotiana species to be
subjected to a curing process. Descriptions of various types of
curing processes for various types of tobaccos are set forth in
Tobacco Production, Chemistry and Technology, Davis et al. (Eds.)
(1999). Exemplary techniques and conditions for curing flue-cured
tobacco are set forth in Nestor et al., Beitrage Tabakforsch. Int.,
20, 467-475 (2003) and U.S. Pat. No. 6,895,974 to Peele, which are
incorporated herein by reference. Representative techniques and
conditions for air curing tobacco are set forth in Roton et al.,
Beitrage Tabakforsch. Int., 21, 305-320 (2005) and Staaf et al.,
Beitrage Tabakforsch. Int., 21, 321-330 (2005), which are
incorporated herein by reference. Certain types of tobaccos can be
subjected to alternative types of curing processes, such as fire
curing or sun curing. Preferably, harvested tobaccos that are cured
are then aged.
At least a portion of the plant of the Nicotiana species (e.g., at
least a portion of the tobacco portion) can be employed in an
immature form. That is, the plant, or at least one portion of that
plant, can be harvested before reaching a stage normally regarded
as ripe or mature. As such, for example, tobacco can be harvested
when the tobacco plant is at the point of a sprout, is commencing
leaf formation, is commencing flowering, or the like. At least a
portion of the plant of the Nicotiana species (e.g., at least a
portion of the tobacco portion) can be employed in a mature form.
That is, the plant, or at least one portion of that plant, can be
harvested when that plant (or plant portion) reaches a point that
is traditionally viewed as being ripe, over-ripe or mature. As
such, for example, through the use of tobacco harvesting techniques
conventionally employed by farmers, Oriental tobacco plants can be
harvested, burley tobacco plants can be harvested, or Virginia
tobacco leaves can be harvested or primed by stalk position.
In accordance with the present invention, a tobacco product
incorporates tobacco that is combined with one or more compounds
extracted and/or isolated from a plant of the Nicotiana species in
green form or cured form. At least a portion of the tobacco product
can comprise compounds removed from the Nicotiana plant (e.g., by
extraction, distillation, or other types of processing techniques).
In some embodiments, at least a portion of the tobacco product can
be composed of degradation products derived from these compounds,
such as compounds collected after subjecting the plants to chemical
reaction or after subjecting compounds or mixtures thereof isolated
from Nicotiana plants to chemical reaction (e.g., acid/base
reaction conditions, oxidation conditions, enzymatic treatment,
and/or heat treatment).
The Nicotiana species can be selected for the content of various
compounds that are present therein. For example, plants can be
selected on the basis that those plants produce relatively high
quantities of one or more of the compounds desired to be isolated
therefrom. In certain embodiments, plants of the Nicotiana species
(e.g., Galpao commun tobacco) are specifically grown for their
abundance of leaf surface compounds. Tobacco plants can be grown in
greenhouses, growth chambers, or outdoors in fields, or grown
hydroponically.
Various parts or portions of the plant of the Nicotiana species can
be employed. For example, virtually all of the plant (e.g., the
whole plant) can be harvested, and employed as such. Alternatively,
various parts or pieces of the plant can be harvested or separated
for further use after harvest. For example, the leaves, stem,
stalk, and various combinations thereof, can be isolated for
further use or treatment. The plant material of the invention may
thus comprise an entire plant or any portion of a plant of the
Nicotiana species.
The post-harvest processing of the plant or portion thereof can
vary. After harvest, the plant or portion thereof can be used in a
green form (e.g., the plant or portion thereof can be used without
being subjected to any curing process). For example, the plant or
portion thereof can be used without being subjected to significant
storage, handling or processing conditions. In certain situations,
it is preferable that the plant or portion thereof be used
virtually immediately after harvest. Alternatively, for example, a
plant or portion thereof in green form can be refrigerated or
frozen for later use, freeze dried, subjected to irradiation,
yellowed, dried, cured (e.g., using air drying techniques or
techniques that employ application of heat), heated or cooked
(e.g., roasted, fried or boiled), or otherwise subjected to storage
or treatment for later use.
The harvested plant or portion thereof can be physically processed.
The plant or portion thereof can be separated into individual parts
or pieces (e.g., the leaves can be removed from the stems, and/or
the stems and leaves can be removed from the stalk. The harvested
plant or individual parts or pieces can be further subdivided into
parts or pieces (e.g., the leaves can be shredded, cut, comminuted,
pulverized, milled or ground into pieces or parts that can be
characterized as filler-type pieces, granules, particulates or fine
powders). The plant, or parts thereof, can be subjected to external
forces or pressure (e.g., by being pressed or subjected to roll
treatment). When carrying out such processing conditions, the plant
or portion thereof can have a moisture content that approximates
its natural moisture content (e.g., its moisture content
immediately upon harvest), a moisture content achieved by adding
moisture to the plant or portion thereof, or a moisture content
that results from the drying of the plant or portion thereof. For
example, powdered, pulverized, ground or milled pieces of plants or
portions thereof can have moisture contents of less than about 25
weight percent, often less than about 20 weight percent, and
frequently less than about 15 weight percent.
The plant of the Nicotiana species or portions thereof can be
subjected to other types of processing conditions. For example,
components can be separated from one another or otherwise
fractionated into chemical classes or mixtures of individual
compounds. Typical separation processes can include one or more
process steps (e.g., solvent extraction using polar solvents,
organic solvents, or supercritical fluids), chromatography,
distillation, filtration, recrystallization, and/or solvent-solvent
partitioning. Exemplary extraction and separation solvents or
carriers include water, alcohols (e.g., methanol or ethanol),
hydrocarbons (e.g., heptane and hexane), diethyl ether, methylene
chloride and supercritical carbon dioxide. Exemplary techniques
useful for extracting components from Nicotiana species are
described in U.S. Pat. No. 4,144,895 to Fiore; U.S. Pat. No.
4,150,677 to Osborne, Jr. et al.; U.S. Pat. No. 4,267,847 to Reid;
U.S. Pat. No. 4,289,147 to Wildman et al.; U.S. Pat. No. 4,351,346
to Brummer et al.; U.S. Pat. No. 4,359,059 to Brummer et al.; U.S.
Pat. No. 4,506,682 to Muller; U.S. Pat. No. 4,589,428 to Keritsis;
U.S. Pat. No. 4,605,016 to Soga et al.; U.S. Pat. No. 4,716,911 to
Poulose et al.; U.S. Pat. No. 4,727,889 to Niven, Jr. et al.; U.S.
Pat. No. 4,887,618 to Bernasek et al.; U.S. Pat. No. 4,941,484 to
Clapp et al.; U.S. Pat. No. 4,967,771 to Fagg et al.; U.S. Pat. No.
4,986,286 to Roberts et al.; U.S. Pat. No. 5,005,593 to Fagg et
al.; U.S. Pat. No. 5,018,540 to Grubbs et al.; U.S. Pat. No.
5,060,669 to White et al.; U.S. Pat. No. 5,065,775 to Fagg; U.S.
Pat. No. 5,074,319 to White et al.; U.S. Pat. No. 5,099,862 to
White et al.; U.S. Pat. No. 5,121,757 to White et al.; U.S. Pat.
No. 5,131,414 to Fagg; U.S. Pat. No. 5,131,415 to Munoz et al.;
U.S. Pat. No. 5,148,819 to Fagg; U.S. Pat. No. 5,197,494 to Kramer;
U.S. Pat. No. 5,230,354 to Smith et al.; U.S. Pat. No. 5,234,008 to
Fagg; U.S. Pat. No. 5,243,999 to Smith; U.S. Pat. No. 5,301,694 to
Raymond et al.; U.S. Pat. No. 5,318,050 to Gonzalez-Parra et al.;
U.S. Pat. No. 5,343,879 to Teague; U.S. Pat. No. 5,360,022 to
Newton; U.S. Pat. No. 5,435,325 to Clapp et al.; U.S. Pat. No.
5,445,169 to Brinkley et al.; U.S. Pat. No. 6,131,584 to
Lauterbach; U.S. Pat. No. 6,298,859 to Kierulff et al.; U.S. Pat.
No. 6,772,767 to Mua et al.; and U.S. Pat. No. 7,337,782 to
Thompson, all of which are incorporated herein by reference. See
also, the types of separation techniques set forth in Brandt et
al., LC-GC Europe, p. 2-5 (March, 2002) and Wellings, A Practical
Handbook of Preparative HPLC (2006), which are incorporated herein
by reference. In addition, the plant or portions thereof can be
subjected to the types of treatments set forth in Ishikawa et al.,
Chem. Pharm. Bull., 50, 501-507 (2002); Tienpont et al., Anal.
Bioanal. Chem., 373, 46-55 (2002); Ochiai, Gerstel Solutions
Worldwide, 6, 17-19 (2006); Coleman, III, et al., J. Sci. Food and
Agric., 84, 1223-1228 (2004); Coleman, III et al., J. Sci. Food and
Agric., 85, 2645-2654 (2005); Pawliszyn, ed., Applications of Solid
Phase Microextraction, RSC Chromatography Monographs, (Royal
Society of Chemistry, UK) (1999); Sahraoui et al., J. Chrom., 1210,
229-233 (2008); and U.S. Pat. No. 5,301,694 to Raymond et al.,
which are incorporated herein by reference.
In particular, in certain embodiments, one or more compounds of
interest are extracted from a plant material of the Nicotiana
species or a portion thereof by contacting the plant or portion
thereof with a solvent under conditions (e.g., suitable pressure
and temperature) sufficient to extract one or more desired
compounds from the plant material. In some embodiments, the solvent
is an organic solvent, such as methanol or hexanes. In other
embodiments, the solvent is dry steam. Dry steam (also referred to
as anhydrous steam) is steam having a minimal content of suspended
water particles (i.e., moisture). For example, dry steam typically
comprises less than about 5% water particles by weight or less than
about 10% water particles by weight.
The temperature and pressure at which the extraction process is
conducted can vary. However, in some embodiments, suitable
extraction is achieved at ambient temperature and pressure.
Further, the amount of time that the solvent is in contact with the
tobacco material can vary. Typically, the solvent will remain in
contact with the tobacco material for approximately two hours,
although longer or shorter time periods can be used without
departing from the invention.
Exemplary compounds of interest that can be present in the extract
obtained according to the methods of the present invention include,
but are not limited to, solanone, neophytadiene,
megastigmatrienone, .beta.-damascenone, norsolanadione,
cis-abienol, .alpha.-cembratrienediol, .beta.-cembratrienediol,
sucrose esters, and/or lutein.
Further processing of the extracted product can be carried out in a
number of ways. The method of further processing can depend on the
compounds present in the extract and/or the type of solvent used in
the extraction.
For example, where an organic solvent (e.g., methanol or hexanes)
is used to extract one or more compounds from the tobacco material,
the solvent brought into contact with the tobacco material can
simply be filtered to remove particulate tobacco material and the
filtrate can be concentrated.
Where dry steam is used to extract one or more compounds from the
tobacco material, the dry steam is typically condensed following
the contacting step to give a steam distillate. In certain
embodiments, this distillation method is conducted on various
mixtures of cured tobacco materials. For example, in one
embodiment, a mixture of flue-cured, burley, and Oriental tobaccos
is used. Methods used for the production of essential oils can be
used herein to distill compounds of interest from tobacco plants or
portions thereof. For exemplary steam distillation processes and
conditions that can be used or modified for use to provide
compounds of interest from tobacco plants or portions thereof
according to the present invention, see for example, U.S. Pat. No.
5,891,501 to McKellip et al., which is incorporated herein by
reference. The type of apparatus used to process the material in
this way can be, for example, the type traditionally employed for
the isolation of peppermint oils. In certain embodiments, the steam
distillate comprises a water condensate layer and an oily layer
that can be separated from each other.
The oily layer thus obtained from the tobacco material typically
comprises one or more compounds of interest (e.g., neophytadiene,
solanone, megastigmatrienone isomers, .beta.-damascenone, and
norsolanadione). In certain embodiments, the oily layer
advantageously is rich in flavor compounds, including one or more
of the compounds of interest noted above, and is essentially
alkaloid free. For example, in certain embodiments, the oily layer
contains less than about 15% by weight, less than about 10% by
weight, less than about 5% by weight, less than about 2% by weight,
less than about 1% by weight, or less than about 0.5% by weight
alkaloids. The percentages of other compounds present in the oily
layer can vary, depending for example, on the type of tobacco
subjected to distillation by the methods provided herein.
The waste water resulting from the distillation process may, in
certain embodiments, also comprise compounds of interest (e.g.,
including, but not limited to, those compounds of interest noted to
be present in the oily layer). These compounds are believed to be
present both in the oily layer and in the water because the
distillation process can provide certain compounds with notable
solubility in water. Therefore, in some embodiments, compounds of
interest are isolated from the waste water produced by the
distillation process. For example, in some embodiments, flavor
compounds and/or nicotine are present in the waste water. It is
noted that the percentage of various volatile and semi-volatile
compounds in the waste water varies as a function of time. Thus,
various fractions of the waste water can be collected separately to
provide solutions containing higher percentages of certain
compounds. Accordingly, the invention provides for the extraction
of desired compounds by distillation, wherein the desired compounds
are provided in the oily layer of the distillate, the water layer
of the distillate, and/or in the waste water produced during the
distillation process.
Various compounds or mixtures of compounds from the Nicotiana plant
or portions thereof can be isolated by the methods provided herein.
As used herein, an "isolated component," or "plant isolate," is a
compound or complex mixture of compounds separated from a plant of
the Nicotiana species or a portion thereof. The isolated component
can be a single compound, a homologous mixture of similar compounds
(e.g., isomers of a flavor compound), or a heterologous mixture of
dissimilar compounds (e.g., a complex mixture of various compounds
of different types, preferably having desirable sensory
attributes). Tobacco material that has been subjected to the
extraction methods described herein may be further processed, e.g.,
to extract one or more additional compounds therefrom. See, for
example, US Patent App. Publ. No. 2008/0254149 to Havkin-Frenkel,
which is incorporated herein by reference.
According to the present invention, a variety of compounds having
distinctive flavor and aroma characteristics can be extracted
and/or isolated from plants of the Nicotiana species. Certain of
those compounds can be considered to be volatile under normal
ambient conditions of temperature, humidity and air pressure.
Preferred compounds exhibit positive sensory attributes at
relatively low concentrations. Examples of the types of compounds
that can be present in Nicotiana plants and extracted and isolated
by the methods described herein include solanone, neophytadiene,
megastigmatrienone, .beta.-damascenone, norsolanadione,
cis-abienol, .alpha.-cembratrienediol, .beta.-cembratrienediol,
sucrose esters, and/or lutein.
Cis-abienol is a major labdanoid in the green leaf of tobaccos. For
example, cis-abienol is commonly found in Oriental tobaccos.
##STR00001##
During air- and sun-curing of green Oriental tobaccos, the
concentration of cis-abienol typically decreases significantly, as
numerous labdanoid degradation products are formed. The labdanoid
degradation products are structurally similar to cis-abienol, and
include, for example, sclareolide, sclareol, and ambroxide. These
degradation products are known to impart cedar characteristics to
tobacco products containing Oriental tobaccos.
##STR00002##
Isolated cis-abienol may find a number of applications in smoking
products and/or smokeless tobacco products. In some embodiments,
isolated cis-abienol is degraded by various means and the
degradation products can be used in smoking products and/or
smokeless tobacco products. For example, in certain embodiments,
isolated cis-abienol and/or degradation products thereof find use
as tobacco-derived, natural flavor materials (e.g., Turkish
replacement flavor material) or Oriental tobacco replacements. In
some embodiments, cis-abienol and/or degradation products are used
for therapeutic or neutriceutical applications. For example,
labdanoid compounds have shown anti-cancer activity. See, for
example, Jung et al., Bioorg. Med. Chem. Lett. 8: 3295-98 (1998),
which is incorporated herein by reference. In some embodiments,
cis-abienol is used as a substrate in the preparation of other
compounds. For example, it has been used as a substrate in the
synthesis of weidendiols, which are cholesterol ester transfer
protein inhibitors that may reduce the risk of atherosclerosis.
See, for example, Barrero et al., Tetrahedron 54: 5635-5650 (1998),
which is incorporated herein by reference.
Cembratrienediols (e.g., .alpha.-2,7,11-cembratriene-4,6-diol and
.beta.-2,7,11-cembratriene-4,6-diol) are found in high quantities
in the leaf and flower of Nicotiana tabacum. Biodegradation of
these compounds during curing of tobacco leaves results in the
formation of a range of flavor compounds. Cembratrienediols have a
structure as depicted below.
##STR00003##
Isolated cembratrienediols may find a number of applications in
smoking products and/or smokeless tobacco products. In some
embodiments, isolated cembratriene diols are degraded by various
means and the degradation products are used in smoking products
and/or smokeless tobacco products. For example, in certain
embodiments, isolated cembratriene diols and/or degradation
products thereof may find use as tobacco-derived, natural flavor
materials. In some embodiments, cembratriene diols and/or
degradation products are used for therapeutic applications. For
example, cembratriene diols have been found to provide potential
health benefits in the treatment of cancer and certain
neurodegenerative diseases. See, for example, Saito et al., Agric.
Biol. Chem. 51(3): 941-43 (1987) and U.S. Pat. No. 4,701,570 to
Mizusaki et al., which are incorporated herein by reference.
Cembratriene diols and derivatives thereof may also be useful as
drugs to prevent smoking and/or to treat nicotine addiction. See,
for example, El Sayed et al., Expert Opin. Invest. Drugs 16(6):
877-87 (2007).
Sucrose esters are glycolipid compounds, characterized by low
molecular weight carboxylic acids attached to hydroxide groups of
the glucose portion of sucrose. Sucrose esters are considered to be
some of the most important aroma and sensory precursors responsible
for Oriental tobacco flavor. See, for example, Leffingwell et al.,
Rec. Adv. Tob. Sci. 14: 169-218 (1998), which is incorporated
herein by reference.
Sucrose esters are typically sucrose molecules comprising three
acyl groups on the glucose ring, each hydrocarbon chain of the acyl
group comprising 3-8 carbon atoms and optionally including one or
more double bonds. The sucrose esters also typically include an
acetyl group on either the glucose ring or the fructose ring, which
gives rise to the common reference to these esters as tetra-acyl
sucrose esters. The exact structure of the sucrose esters isolated
according to the present invention can vary somewhat as to
placement, chain length, and saturation of the acyl groups, most
sucrose esters of Oriental tobacco are encompassed by the following
structure:
##STR00004##
wherein each R is an independently selected C3-C8 hydrocarbon,
which can be straight or branched and saturated or unsaturated, and
both R.sub.1 substituents are H or one R.sub.1 is H and the other
is acetyl (--C(O)CH.sub.3). Most common R groups comprise butyl,
pentyl, and hexyl.
Lutein is a major carotenoid pigment in tobacco.
##STR00005##
Lutein is known to break down during the air-curing of green
tobacco to produce several ionones and derivatives thereof. Two
important types of derivatives of lutein are megastigmatrienones
and beta-damascenone. These compounds impact the aroma
characteristics of cured tobacco. In addition to being degradation
products of lutein, megastigmatrienone and beta-damascenone may
also be separately extracted and isolated from tobacco according to
the methods provided herein.
##STR00006##
Isolated lutein may find a number of applications in smoking
products and/or smokeless tobacco products. In some embodiments,
isolated lutein is degraded by various means and the degradation
products produced therefrom can be used in smoking products and/or
smokeless tobacco products. For example, in certain embodiments,
isolated lutein and/or degradation products thereof may find use as
tobacco-derived, natural flavor materials, colorants, or
antioxidants. Lutein is also useful as a nutritional and/or
therapeutic compound. See for example, Granado et al., Br. J. Nutr.
90(3): 487-502 (2003); Sies et al., Int. J. Vitam. Nutr. Res.
73(2): 95-100 (2003); and Krinsky et al., Annu. Rev. Nutr. 23(2):
171-201 (2003), which are incorporated herein by reference.
Solanone is a compound that is a useful tobacco flavorant and
flavor enhancer. It is specifically noted to be present in burley
tobacco aroma. See, for example, Domle et al., Helv. Chim. Acta,
55(6): 1866-1882 (1972), which is incorporated herein by reference.
Although it is commonly produced by synthetic means (see, e.g.,
Johnson et al., J. Org. Chem. 30(9): 2918-2921 (1965); and U.S.
Pat. No. 4,433,695 to Hall et al. and U.S. Pat. No. 4,547,594 to
Light et al., which are incorporated herein by reference), solanone
is present in tobacco and may be isolated according to the methods
provided herein.
##STR00007##
Isolated solanone may find a number of applications in smoking
products and/or smokeless tobacco products. In some embodiments,
isolated solanone may be degraded by various means and the
degradation products may be used in smoking products and/or
smokeless tobacco products. For example, in certain embodiments,
isolated solanone and/or degradation products thereof may find use
as tobacco-derived, natural flavor materials.
Neophytadiene is reported to be a tobacco flavor enhancer in that
it may act as a flavor carrier by entrapping volatiles in the
tobacco smoke aerosol. See, for example, J. C. Leffingwell, Leaf
chemistry: basic chemical constituents of tobacco leaf and
differences among tobacco types. Tobacco: Production, Chemistry and
Technology, 265-284 (1999).
##STR00008##
Isolated neophytadiene may find a number of applications in smoking
products and/or smokeless tobacco products. In some embodiments,
isolated neophytadiene may be degraded by various means and the
degradation products may be used in smoking products and/or
smokeless tobacco products. For example, in certain embodiments,
isolated neophytadiene and/or degradation products thereof may find
use as tobacco-derived, natural flavor materials.
Norsolanadione is another compound that is known to be useful as a
tobacco flavorant and in augmenting or enhancing the aroma and
taste of smoking tobacco. Like solanone, this compound is commonly
synthesized, rather than isolated. See, for example, U.S. Pat. No.
4,517,385 to Light et al., which is incorporated herein by
reference.
##STR00009##
Isolated norsolanadione may find a number of applications in
smoking products and/or smokeless tobacco products. In some
embodiments, isolated norsolanadione may be degraded by various
means and the degradation products may be used in tobacco products.
For example, in certain embodiments, isolated norsolanadione and/or
degradation products thereof may find use as tobacco-derived,
natural flavor materials.
Following extraction of compounds of interest from tobacco, it is
desirable according to the present invention to further isolate and
purify certain compounds. Because the extraction processes
disclosed herein typically lead to complex mixtures of compounds,
they must be further treated to provide desired mixtures of
compounds and/or single isolated compounds (e.g., to give isolates
comprising at least about 75% by weight of the compound or
compounds).
The means by which such mixtures and/or single isolated compounds
are provided can vary. Additional solvent extractions (e.g.,
solvent extraction using polar solvents, organic solvents, or
supercritical fluids), chromatography, distillation, filtration,
recrystallization, and/or solvent-solvent partitioning may be used
to isolate and/or purify desired compounds from the extracts.
In some embodiments, multiple methods are used to isolate and/or
purify the desired compounds. For example, solvent extraction may
be combined with one or more chromatographic methods. The sample
obtained via extraction may be dissolved in a solvent and injected
directly onto the flash chromatography system or may be treated in
some way prior to injection. In another example, in some
embodiments, the sample is first treated to remove one or more
compounds that are known to elute under similar conditions as the
compound(s) to be isolated by flash chromatography. In one
particular embodiment, an extract obtained by methanol extraction
of a tobacco material is processed to remove quercetin-3-rutinoside
("rutin"). For example, the rutin can be removed from the extract
by adding water, methanol, and methylene chloride to the extract
and extracting the rutin into the methanol/water layer. The
methylene chloride layer can be concentrated and further processed
(e.g., by chromatography) to isolate and/or purify the desired
compound(s) contained therein. In other embodiments, the extract
can be dissolved and directly subjected to chromatographic
separation.
In some embodiments, preparative liquid chromatography is used to
isolate and/or purify certain compounds of interest from a tobacco
extract. In some embodiments, a compound or compounds of interest
are isolated using preparative liquid chromatography based on the
elution times of standards. Various automated commercial prep-LC
systems are available, from manufacturers including Waters, Agilent
Technologies, and Bio-Rad. The specific parameters of the prep LC
system used can be varied by one of skill in the art to achieve the
desired level of resolution. For example, the solvent may be any
solvent or mixture of solvents sufficient to dissolve the
compound(s) of interest. The solvent may be, for example, water,
methanol, ethanol, ethyl acetate, diethyl ether, methylene
chloride, chloroform, petroleum ether, and/or hexanes. The system
may be operated with an isocratic or gradient solvent system (i.e.,
varying the ratio of two or more solvents as a function of time).
In some embodiments, the solvent system can be chosen such that it
provides the best resolution between the compound of interest and
other compounds present in the mixture. The flow rate of the system
may be varied, for example, from about 10 mL/min to about 100
mL/min (e.g., about 36 mL/min).
In some embodiments, flash chromatography is used to isolate and/or
purify certain compounds of interest from a tobacco extract. Flash
chromatography systems are known in the art and exemplary systems
are discussed, for example, in Still et al., J. Org. Chem. 42:
2923-2925 (1978) and U.S. Pat. No. 4,591,442 to Andrews, which are
incorporated herein by reference. Various automated commercial
flash chromatography systems are available, from manufacturers
including Biotage, Teledyne Isco, Grace Davison Discovery Sciences,
and Buchi. Flash chromatography may be desirable to provide
reasonably large quantities of compound, as columns typically have
relatively large particle sizes (e.g., roughly 30-40 .mu.m) and can
accommodate a greater quantity of sample (and a more concentrated
sample), allowing more of the compound(s) of interest to be
isolated per injection.
The specific parameters of the flash chromatography system used can
be varied by one of skill in the art to achieve the desired level
of resolution. For example, the solvent may be any solvent or
mixture of solvents sufficient to dissolve the compound(s) of
interest. The solvent may be, for example, water, methanol,
ethanol, ethyl acetate, diethyl ether, methylene chloride,
chloroform, petroleum ether, and/or hexanes. The system may be
operated with an isocratic or gradient solvent system (i.e.,
varying the ratio of two or more solvents as a function of time).
In some embodiments, the solvent system may be chosen to provide
the best resolution between the compound of interest and other
compounds present in the mixture. The flow rate of the system may
be varied, for example, from about 20 to about 200 mL/min (e.g.,
about 150 mL/min).
Flash chromatography may or may not provide the compound(s) of
interest at a sufficient purity level. In certain embodiments, the
fractions corresponding to the compound(s) of interest may be
collected, combined, and concentrated to give an isolate comprising
the compound(s) of interest at a sufficient level of purity (i.e.,
wherein the compound(s) of interest are present in a sufficient
weight percentage of the isolate). For example, the isolate of the
present invention can comprise the compound(s) of interest in an
amount of greater than about 75% by weight, greater than about 80%
by weight, greater than about 85% by weight, greater than about 90%
by weight, greater than about 95% by weight, greater than about 98%
by weight, or greater than about 99% by weight. In some
embodiments, fractions obtained from flash chromatography can be
further resolved using preparative liquid chromatography.
In some embodiments, isolated compounds or mixtures thereof can be
subjected to conditions so as to cause those compounds to undergo
chemical transformation. For example, the tobacco material obtained
from plants of the Nicotiana species or portion thereof can be
treated to cause chemical transformation or be admixed with other
ingredients. In some embodiments, the extracts obtained therefrom,
or the isolated compound(s) (isolates) can be treated to cause
chemical transformation or be admixed with other ingredients. The
chemical transformations or modification of the tobacco material,
extract, or isolated compound can result in changes of certain
chemical and physical properties of the tobacco material, extract,
or isolated compound(s) (e.g., the sensory attributes thereof).
Exemplary chemical modification processes can be carried out by
acid/base reaction, hydrolysis, oxidation, heating and/or enzymatic
treatments; and as such, compounds can undergo various degradation
reactions.
In certain embodiments, the tobacco material, extract, or isolate
is treated to provide degradation products (e.g., lutein may be
treated to provide various flavor compounds, including
megastigmatrienones and/or .beta.-damascenone; cis-abienol may be
treated to provide sclareolide, sclareol, and/or ambroxide).
Degradation products are any compounds that are produced from the
compounds extracted and/or isolated according to the present
invention. Degradation products can be formed naturally from such
compounds or may be produced by an accelerated degradation process
(e.g., by the addition of heat and/or chemicals to accelerate the
breakdown of the compounds). These compounds can be degraded, for
example, by means of oxidation (e.g., through treatment with
H.sub.2O.sub.2 or other oxidizing agents) and/or hydrolysis
reactions.
Exemplary types of further ingredients that can be admixed with the
tobacco material, extracts, or isolates according to the present
invention include one or more flavorants, fillers, binders, pH
adjusters, buffering agents, colorants, disintegration aids,
antioxidants, humectants and preservatives.
The extracts and isolates of the present invention are useful as
components added to tobacco compositions, particularly tobacco
compositions incorporated into smoking articles or smokeless
tobacco products. Addition of the extracts or isolates to a tobacco
composition can enhance the tobacco composition in a variety of
ways, depending on the nature of the extract or isolate and the
type of tobacco composition. Exemplary extracts and isolates can
serve to provide flavor and/or aroma to a tobacco product (e.g.,
composition that alters the sensory characteristics of tobacco
compositions or smoke derived therefrom). The extracts and isolates
of the invention can also be used as components of tobacco products
that contain no other tobacco material therein. In other words, the
extract or isolate of the invention could be used as the sole
source of tobacco in the tobacco product of the invention by, for
example, incorporating the extract or isolate into an oral
smokeless tobacco composition, such as a product adapted to
dissolve or melt in the oral cavity.
The form of the extract or isolate obtained according to the
present invention can vary. Typically, the isolate is in a solid,
liquid, or semi-solid or gel form. The isolate can be used in
concrete, absolute, or neat form. Solid forms of the isolate
include spray-dried and freeze-dried forms. Liquid forms of the
isolate include isolate contained within aqueous or organic solvent
carriers.
The extract or isolate can be employed as a component of a tobacco
composition in a variety of ways. The extract or isolate can be
employed as a component of processed tobaccos. In one regard, the
extract or isolate can be employed within a casing formulation for
application to tobacco strip (e.g., using the types of manners and
methods set forth in U.S. Pat. No. 4,819,668 to Shelar, which is
incorporated herein by reference) or within a top dressing
formulation. Alternatively, the extract or isolate can be employed
as an ingredient of a reconstituted tobacco material (e.g., using
the types of tobacco reconstitution processes generally set forth
in U.S. Pat. No. 5,143,097 to Sohn; U.S. Pat. No. 5,159,942 to
Brinkley et al.; U.S. Pat. No. 5,598,868 to Jakob; U.S. Pat. No.
5,715,844 to Young; U.S. Pat. No. 5,724,998 to Gellatly; and U.S.
Pat. No. 6,216,706 to Kumar, which are incorporated herein by
reference). The extract or isolate also can be incorporated into a
cigarette filter (e.g., in the filter plug, plug wrap, or tipping
paper) or incorporated into cigarette wrapping paper, preferably on
the inside surface, during the cigarette manufacturing process.
The Nicotiana-derived extract or isolate can be incorporated into
smoking articles. Representative tobacco blends, non-tobacco
components, and representative cigarettes manufactured therefrom,
are set forth in U.S. Pat. No. 4,836,224 to Lawson et al.; U.S.
Pat. No. 4,924,888 to Perfetti et al.; U.S. Pat. No. 5,056,537 to
Brown et al.; U.S. Pat. No. 5,220,930 to Gentry; and U.S. Pat. No.
5,360,023 to Blakley et al.; US Pat. Application 2002/0000235 to
Shafer et al.; and PCT WO 02/37990. Those tobacco materials also
can be employed for the manufacture of those types of cigarettes
that are described in U.S. Pat. No. 4,793,365 to Sensabaugh; U.S.
Pat. No. 4,917,128 to Clearman et al.; U.S. Pat. No. 4,947,974 to
Brooks et al.; U.S. Pat. No. 4,961,438 to Korte; U.S. Pat. No.
4,920,990 to Lawrence et al.; U.S. Pat. No. 5,033,483 to Clearman
et al.; U.S. Pat. No. 5,074,321 to Gentry et al.; U.S. Pat. No.
5,105,835 to Drewett et al.; U.S. Pat. No. 5,178,167 to Riggs et
al.; U.S. Pat. No. 5,183,062 to Clearman et al.; U.S. Pat. No.
5,211,684 to Shannon et al.; U.S. Pat. No. 5,247,949 to Deevi et
al.; U.S. Pat. No. 5,551,451 to Riggs et al.; U.S. Pat. No.
5,285,798 to Banerjee et al.; U.S. Pat. No. 5,593,792 to Farrier et
al.; U.S. Pat. No. 5,595,577 to Bensalem et al.; U.S. Pat. No.
5,816,263 to Counts et al.; U.S. Pat. No. 5,819,751 to Barnes et
al.; U.S. Pat. No. 6,095,153 to Beven et al.; U.S. Pat. No.
6,311,694 to Nichols et al.; and U.S. Pat. No. 6,367,481 to
Nichols, et al.; US Pat. Appl. Pub. No. 2008/0092912 to Robinson et
al.; and PCT WO 97/48294 and PCT WO 98/16125. See, also, those
types of commercially marketed cigarettes described Chemical and
Biological Studies on New Cigarette Prototypes that Heat Instead of
Burn Tobacco, R.J. Reynolds Tobacco Company Monograph (1988) and
Inhalation Toxicology, 12:5, p. 1-58 (2000).
The extract or isolate described herein can be incorporated into
smokeless tobacco products, such as loose moist snuff, loose dry
snuff, chewing tobacco, pelletized tobacco pieces (e.g., having the
shapes of pills, tablets, spheres, coins, beads, obloids or beans),
extruded or formed tobacco strips, pieces, rods, cylinders or
sticks, finely divided ground powders, finely divided or milled
agglomerates of powdered pieces and components, flake-like pieces,
molded processed tobacco pieces, pieces of tobacco-containing gum,
rolls of tape-like films, readily water-dissolvable or
water-dispersible films or strips (e.g., US Pat. App. Pub. No.
2006/0198873 to Chan et al.), or capsule-like materials possessing
an outer shell (e.g., a pliable or hard outer shell that can be
clear, colorless, translucent or highly colored in nature) and an
inner region possessing tobacco or tobacco flavor (e.g., a
Newtoniam fluid or a thixotropic fluid incorporating tobacco of
some form). Various types of smokeless tobacco products are set
forth in U.S. Pat. No. 1,376,586 to Schwartz; U.S. Pat. No.
3,696,917 to Levi; U.S. Pat. No. 4,513,756 to Pittman et al.; U.S.
Pat. No. 4,528,993 to Sensabaugh, Jr. et al.; U.S. Pat. No.
4,624,269 to Story et al.; U.S. Pat. No. 4,987,907 to Townsend;
U.S. Pat. No. 5,092,352 to Sprinkle, III et al.; and U.S. Pat. No.
5,387,416 to White et al.; US Pat. App. Pub. Nos. 2005/0244521 to
Strickland et al. and 2008/0196730 to Engstrom et al.; PCT WO
04/095959 to Arnarp et al.; PCT WO 05/063060 to Atchley et al.; PCT
WO 05/016036 to Bjorkholm; and PCT WO 05/041699 to Quinter et al.,
each of which is incorporated herein by reference. See also, the
types of smokeless tobacco formulations, ingredients, and
processing methodologies set forth in U.S. Pat. No. 6,953,040 to
Atchley et al. and U.S. Pat. No. 7,032,601 to Atchley et al.; US
Pat. Appl. Pub. Nos. 2002/0162562 to Williams; 2002/0162563 to
Williams; 2003/0070687 to Atchley et al.; 2004/0020503 to Williams,
2005/0178398 to Breslin et al.; 2006/0191548 to Strickland et al.;
2007/0062549 to Holton, Jr. et al.; 2007/0186941 to Holton, Jr. et
al.; 2007/0186942 to Strickland et al.; 2008/0029110 to Dube et
al.; 2008/0029116 to Robinson et al.; 2008/0029117 to Mua et al.;
2008/0173317 to Robinson et al.; and 2008/0209586 to Neilsen et
al., each of which is incorporated herein by reference.
The amount of extract or isolate added to a tobacco composition, or
otherwise incorporated within a tobacco composition or tobacco
product, can depend on the desired function of that extract or
isolate, the chemical makeup of that extract or isolate, and the
type of tobacco composition to which the extract or isolate is
added. The amount added to a tobacco composition can vary, but will
typically range from about 5 ppm to about 5 weight percent based on
the total dry weight of the tobacco composition to which the
extract or isolate is added. The amount added may vary, depending,
for example, on the goal to be achieved by addition of such
compound or mixture of compounds (e.g., the enhancement of flavor)
and on the specific characteristics of the compound or mixture of
compounds to be added.
EXPERIMENTAL
Aspects of the present invention is more fully illustrated by the
following examples, which are set forth to illustrate certain
aspects of the present invention and are not to be construed as
limiting thereof.
Example 1
Extraction and Isolation of Sucrose Esters, Lutein,
Cembratrienediols and Cis-Abienol
Extraction
Nicotiana tabacum Galpao tobacco is harvested, chopped into pieces,
and extracted with methanol. The leaves are removed and the
methanol extract is concentrated to approximately 77% solids by
weight. The concentrated methanol extract separates into two
layers, comprising an upper tar-like layer (which is soluble in
methanol) and a lower aqueous layer (which is soluble in water).
The upper tar-like layer is separated and is found to contain leaf
surface analytes of interest such as sucrose esters,
cembratrienediols, and cis-abienol. These analytes of interest can
be separated and collected by liquid chromatography and/or flash
chromatography.
Isolation Methods
Preparative scale liquid chromatography is commonly used to
separate and collect compounds of interest in a complex mixture
(e.g., using a Waters prep LC system comprising a Waters 2707
Autosampler, a Waters DAD detector, a Waters 2545 Quaternary
Gradient Module, and a Waters Fraction Collector III and equipped
with a Waters .mu.Bondapak C18 19.times.300 mm column and fraction
collector).
Separation methods are typically developed on an analytical scale
liquid chromatography column (e.g., using a Waters 2695 LC system
equipped with a Waters .mu.Bondapak C18 3.9.times.300 mm 10 .mu.m
particle column) and scaled up to a preparative scale liquid
chromatography system. Flash chromatography (e.g., using a Teledyne
Isco CombiFlash Automated Flash Purification System with 275 g C18
Gold column) may also be used to isolate compounds of interest.
This technique can be used alone or in tandem with preparative
chromatography to isolate and collect various compounds.
Isolation of Sucrose Esters
Sucrose esters are isolated from the tar-like layer by preparative
scale liquid chromatography. The tar-like mixture is dissolved in
methanol (roughly 20 mg/mL) and injected (1 mL) on the Waters prep
LC system noted above, operated at a flow rate of 36 mL/min,
isocratic analysis with 75:25 methanol: water, and a Waters DAD
detector at 214 nm. The fractions corresponding to the known
retention time of sucrose ester standards (collected between about
1.0 and about 5.6 minutes) are collected and combined. Another
major peak is noticed to elute with the sucrose esters at
approximately 1.6 minutes. HR-LC-MS analysis of these fractions
shows that this peak corresponds to quercetin-3-rutinoside or
rutin. The concentration of rutin in the tar-like mixture as
determined by HR-LC-MS analysis is approximately 800 .mu.g/g.
To generate an extract free of the rutinoside, a liquid-liquid
extraction is performed on the tar-like layer prior to prep LC. The
tar-like layer (about 2 g) is dissolved in methanol (30 mL). This
mixture is added to a reparatory funnel with distilled, deionized
water (80 mL) and methylene chloride (40 mL). The mixture is
shaken, the aqueous layer (shown to contain the rutin) is
discarded, and the methylene chloride layer is removed and
concentrated. The resulting material (reconstituted in methanol) is
analyzed by HR-LC-MS, which showed the presence of sucrose esters
but not the rutinoside. The rutinoside-free material is then
injected onto the prep LC system to isolate the sucrose esters.
The fractions corresponding to the known retention time of sucrose
ester standards are collected and combined. The combined fractions
are concentrated to remove methanol and methylene chloride is added
to the remaining water layer in a 1:1 ratio. The tubes are shaken,
centrifuged, and the aqueous layer is discarded. The methylene
chloride layer is concentrated and dissolved in isopropyl alcohol
for HR-LC-MS analysis. The tar-like layer of tobacco methanol
extract is determined to contain approximately 1,000 .mu.g/g of a
range of sucrose esters. This extract exhibits a similar
qualitative distribution of sucrose esters as is present in other
Oriental, cured tobacco types.
Isolation of Lutein
Lutein is isolated by prep LC from the tar-like layer, which is
first treated to remove rutinoside, as described above. The
rutinoside-free mixture is injected onto the prep LC system (10 mL
injection volume) comprising a Symmetry Prep C18 19.times.300 mm 7
.mu.m particle column at ambient temperature, operated at a flow
rate of 26 mL/min, a solvent system of methanol and water, with
initial ratio of 75:25, ratio at 10 minutes of 75:25, ratio at 15
minutes of 100:0, and ratio at 25 minutes of 75:25: water, and a
Waters DAD detector at 443 nm. The fraction collector is set to
collect 40 second fractions throughout the run, with total analysis
time of 30 minutes. Lutein elutes at approximately 18.7 minutes
under these conditions (as correlated with a lutein standard
previously injected onto the prep LC system).
Lutein is also isolated from the tar-like layer by flash
chromatography. A sample of the tar-like layer, which is first
treated to remove rutinoside, as described above, is injected (15
mL injection volume) onto the flash chromatography system. The
flash chromatography system is operated at a flow rate of 150
mL/min with a solvent (methanol and water) gradient with initial
ratio of 75:25, ratio at 5 minutes of 75:25, ratio at 7 minutes of
100:0, and ratio at 15 minutes of 75:25, and a detector set at 443
nm. The fractions giving rise to a signal at 443 nm after the
elution of cis-abienol were collected on the flash chromatography
system, combined, and concentrated (e.g., using a Buchi Rotavapor
unit set at 50.degree. C. and a vacuum of 337 mBar). The combined,
concentrated fractions are dissolved and injected onto the prep LC
system (10 mL injection volume) comprising a Symmetry Prep C18
19.times.300 mm 7 .mu.m particle column at ambient temperature,
operated at a flow rate of 26 mL/min, a solvent system of methanol
and water, with initial ratio of 75:25, ratio at 3 minutes of
75:25, ratio at 5 minutes of 100:0, and ratio at 12 minutes of
75:25: water, and a Waters DAD detector at 443 nm. The fraction
collector is set to collect 20 second fractions throughout the run,
with total analysis time of 15 minutes. Lutein elutes at 9
minutes.
Lutein-containing fractions that have been isolated by prep LC
and/or flash chromatography are combined and concentrated to remove
methanol (e.g., using a Buchi Rotovapor unit set at 50.degree. C.
and a vacuum of 337 mBar). The resulting material is a semi-solid
form of lutein.
Isolation of Cembratrienediols
Cembratrienediols are isolated from the tar-like layer by
preparative scale liquid chromatography. A sample of the tar-like
layer described above, is injected (1 mL injection volume) onto the
flash chromatography system. The flash chromatography system is
operated at a flow rate of 36 mL/min with an isocratic solvent
system (75:25 methanol:water) and a detector set at 214 nm. The
fractions giving rise to a signal at 214 nm were collected on the
flash chromatography system. The fraction collector is set to
collect 20 second fractions throughout the run, with total analysis
time of 10 minutes. Using the retention time of
.beta.-cembratrienediol and .alpha.-cembratrienediol standards, the
fractions corresponding to each of these compounds were collected,
and separately combined. On this system, the
.beta.-cembratrienediol is eluted at 9.0 minutes and the
.alpha.-cembratrienediol is eluted at 6.9 minutes.
The isolated fractions are separately concentrated to remove
methanol from the fractions, leaving aqueous solutions. Methylene
chloride is added to each isolated fraction, the fractions are
shaken to isolate the desired compound in the methylene chloride
layer, and the fractions are centrifuged. The aqueous layer is
discarded and the remaining methylene chloride layers are analyzed
by GC/MS (e.g., using an Agilent 6890/5973 system from Agilent).
GC/MS is conducted by adding DMF with internal standard (400 ppm
tert-butyl hydroquinone) and BSTFA with 1% TMCS to vials containing
the isolated fractions. The vials are kept at 76.degree. C. for 30
minutes and cooled to room temperature for 30 minutes. The
resulting solutions are analyzed by GC/MS, and peak identification
is done by comparing the spectra of the silylated cembratrienediols
with the spectra of known standards.
The mass spectra confirms that the combined fractions are the
.alpha. and .beta. cembratriene diols, and the data indicates that
the both cembratrienediol fractions are approximately 99% pure
based on the total area count. The .beta.-cembratrienediol fraction
contains a small amount of sugar-like compounds and hexadecanoic
acid and the .alpha.-cembratrienediol fraction contains small
amounts of sugar-like compounds and a phytol-like compound. The
tar-like layer of tobacco methanol extract is determined to contain
approximately 80 mg/g .beta.-cembratrienediol and approximately 30
mg/g .alpha.-cembratrienediol.
Isolation of Cis-Abienol
Cis-abienol is isolated from the tar-like layer by preparative
scale liquid chromatography. A sample of the tar-like layer is
first treated to remove rutinoside, as described above. The
rutinoside-free mixture is injected onto the prep LC system (10 mL
injection volume) comprising a Symmetry Prep C18 19.times.300 mm 10
.mu.m particle column at ambient temperature, operated at a flow
rate of 36 mL/min, an isocratic solvent system of methanol and
water, with ratio of 75:25, and a Waters DAD detector at 214 nm.
The fraction collector is set to collect 20 second fractions
throughout the run (of 12 mL each), with total analysis time of 20
minutes. Cis-abienol elutes at approximately 17.0 minutes under
these conditions.
Initially, the isolated fractions with a significant absorption
peak at 214 nm, are unknown. Thus, the combined fractions are
analyzed by GC/MS, which is inconclusive. The isolated fraction is
infused onto the Thermo TSQ Quantum Ultra MS/MS using an
Atmospheric Pressure Chemical Ionization probe for ionization of
the sample. This technique indicates that the molecular weight of
the compound in the isolated fraction is 29 amu. The isolated
fraction is additionally analyzed by UV/Vis spectroscopy (e.g.,
using a Hewlett Packard 8453 UV/Vis spectrophotometer), which shows
a maximum absorption at 238 nm. Further confirmation of the
identity of this isolated fraction is based on reported HPLC-ESI-MS
molecular weight, NMR, and UV/Vis data on cis-abienol in an article
by Ding et al., Chromatographia 66:529-532 (2007), which is
incorporated herein by reference.
Cis-abienol is alternatively isolated from the tar-like layer by
flash chromatography. A sample of the tar-like layer, which is
first treated to remove rutinoside, as described above, is injected
(15 mL injection volume) onto the flash chromatography system. The
flash chromatography system is operated at a flow rate of 150
mL/min with a solvent (methanol and water) gradient with initial
ratio of 75:25, ratio at 5 minutes of 75:25, ratio at 7 minutes of
100:0, and ratio at 15 minutes of 75:25, and a detector set at 214
nm. The fractions giving rise to a signal at 214 nm are collected
on the flash chromatography system, combined, and concentrated
(e.g., using a Buchi Rotavapor unit set at 50.degree. C. and a
vacuum of 337 mBar). The combined, concentrated fractions are
dissolved and injected onto the prep LC system (10 mL injection
volume) comprising a Symmetry Prep C18 19.times.300 mm 7 .mu.m
particle column at ambient temperature, operated at a flow rate of
26 mL/min, a solvent system of methanol and water, with initial
ratio of 75:25, ratio at 10 minutes of 75:25, ratio at 15 minutes
of 100:0, and ratio at 20 minutes of 75:25, and a Waters DAD
detector at 214 nm. The fraction collector is set to collect 20
second fractions throughout the run (12 mL volume of each), with
total analysis time of 30 minutes. Cis-abienol elutes at 17.8
minutes.
The cis-abienol fractions collected from the preparative LC and/or
the flash chromatography method are concentrated to provide a
semi-solid form of cis-abienol. High volume isolation and
collection of cis-abienol are typically conducted on the flash
chromatography system, which allows for greater efficiency, as more
concentrated samples can be injected. In some cases, fractions from
flash chromatography must be further resolved using preparative
HPLC; however, flash chromatography typically provides sufficient
resolution of cis-abienol without the need for preparative HPLC of
the fractions.
Example 2
Extraction and Isolation of Sucrose Esters, Lutein,
Cembratrienediols and Cis-Abienol
A mixture of flue-cured, burley, and Oriental tobaccos is subjected
to a dry steam distillation. Specifically, a strip blend of flue
cured, burley, and Oriental tobaccos is placed in a 640 ft.sup.3
wagon equipped with steam distillation capability Anhydrous steam
is passed through the wagon and condensed, producing approximately
4 gallons per minute of steam distillate. The distillate is
processed employing equipment traditionally employed for the
isolation of peppermint oils. A few minutes after beginning the
steam distillation process, an oil sheen begins to appear on the
surface of the collected distillate. As time progresses, the sheen
becomes a defined oil layer resting on top of the water condensate.
This reddish-brown essential oil is gently removed from the
water.
The essential oil is dissolved in methylene chloride and analyzed
by gas chromatography/mass spectrometry (GC/MS, e.g., Agilent 6890
GC equipped with Agilent 5973 MSD). Data indicates that the
essential oil comprises, as major volatile and semi-volatile
compounds, solanone, neophytadiene, palmitic acid, and oleic acid.
Other components of the essential oil are megastigmatrienone
isomers, ionol derivatives, .beta.-damascenone, and
norsolanadione.
Further, the distillate water that was first passed through the
essential oil isolation equipment and subsequently exhausted to the
sewer (i.e., the "waste" stream) is captured. Samples of the
"waste" stream collected during the distillation as a function of
time are dissolved in methylene chloride and analyzed by GC/MS. The
resulting chromatograms indicate that the "waste" stream contains a
notable number of volatile and semi-volatile compounds including
some of the compounds found in the essential oil (e.g.,
neophytadiene, nicotine, furfuryl alcohol, and bipyridine).
The "waste" stream is separated into aqueous and organic components
to facilitate downstream processing/separation of the essential oil
into less complex mixtures or individual components. Specifically,
"waste" water from the steam distillation process is added to
silica contained within a fritted glass cylinder. The water is
gently removed from the silica using a water aspirator vacuum.
Hexane is percolated through the column, followed by MTBE, followed
by methanol. The organic solvents are removed by rotary evaporation
and the resulting materials are reconstituted in methylene
chloride. The methylene chloride samples are analyzed by GC/MS. The
major component of the hexane extract is neophytadiene.
Example 3
Extraction and Isolation of Sucrose Esters and
Cembratrienediols
A tobacco leaf is dipped into methylene chloride at room
temperature for approximately 30 seconds. The resulting methylene
chloride solution is concentrated and the extract is cleaned using
a method described in Ashraf-Khorassani et al., Beitrage
Tabkforsch. Int. 23: 32-45 (2008), which is incorporated herein by
reference in its entirety.
The cleaned extract is purified with normal phase HPLC with UV
detection at 214 nm, using a cyano-bonded silica column (25
cm.times.10 mm, d.sub.p=5 .mu.m) and a mobile phase of
ethanol/iso-octane/water in a ratio of 15:85:0.1. Peaks are
identified for sucrose esters, .alpha.-cembrene diol and
.beta.-cembrene diol; fractions corresponding to each peak are
collected individually. The identities of the fractions
corresponding to each peak are confirmed by GC-FID (MS), DB5, 15
m.times.0.25 mm (TMS derivatives) with an initial oven temperature
of 80.degree. C. (held for 2 minutes), which was ramped up
10.degree. C./min to 140.degree. C., ramped up 5.degree. C./min to
300.degree. C. and held at 300.degree. C. for 10 minutes.
Many modifications and other embodiments of the invention will come
to mind to one skilled in the art to which this invention pertains
having the benefit of the teachings presented in the foregoing
description. Therefore, it is to be understood that the invention
is not to be limited to the specific embodiments disclosed and that
modifications and other embodiments are intended to be included
within the scope of the appended claims. Although specific terms
are employed herein, they are used in a generic and descriptive
sense only and not for purposes of limitation.
* * * * *